Covalent attachment of a photoremoveable group to a parent compound (i.e. "caging") to alter its physical or biological properties has been exploited extensively for following components of dynamic systems. As used herein, the term "cage" refers to a photolytically sensitive substituent that is designed to interfere with the reactivity or other physical properties of the free probe. Photolysis (typically by illumination in the UV (250-400 nm) region of the spectrum) cleaves the caging group from the parent compound, restoring its normal properties. In this way it is possible to release the parent compound into the system of interest with much better temporal and spatial resolution than is possible by simple diffusion.
Appropriate caging groups for compounds used in the study of processes that change rapidly, such as biological processes, must be photolyzed rapidly and with relatively high quantum yield. It is also important that caging alters some property of the parent compound to the desired level, and that the caged compound remains useful in the system of interest. A variety of caged probes exist. For example, the o-nitrobenzyl group has been used to cage the calcium ion mobilizer 1,4,5-inositol triphosphate (IP.sub.3). However, IP.sub.3 acts by mobilizing intracellular calcium stores, and does not itself transport calcium ions.
Calcium ion levels in cells have been manipulated using other caged probes. Both diazo-2 and caged EGTA have been used to alter physiological calcium levels in cells. Nitrophenyl EGTA is a photolabile Ca.sup.2+ chelator that exhibits an .about.12,500-fold decrease in binding affinity upon photolysis. Therefore, upon illumination of nitrophenyl-EGTA, the calcium ions bound to the chelator are released, increasing cytoplasmic Ca.sup.2+ levels (U.S. Pat. No.5,446,186 to Ellis-Davies et al. (1995)). Similarly, 4,5-dimethoxy-2-nitrophenyl EDTA (sold under the trademark DM-NITROPHEN) releases calcium ions upon illumination (U.S. Pat. No. 4,981,985 to Kaplan et al. (1991)). In contrast, diazo-2 is a photolabile calcium indicator whose affinity for Ca.sup.2+ increases approximately 30-fold upon illumination. Diazo-2 has been used to rapidly decrease cytoplasmic Ca.sup.2+ levels in tensed frog muscle cells and in rat fibroblasts (Diazo-2 is described in U.S. Pat. No. 5,141,627 to Tsien et al. (1992)). While these caged probes are useful for manipulating intracellular calcium ion levels, they function by physically depleting or introducing the ions themselves from within the intracellular milieu. The caged probes of the present invention, in contrast, function by actively shuttling ions across cell membranes or other semi-permeable barriers after photolysis of the caging group.
The present invention describes caged ionophores that can be used to study the physiological effects of the free ionophore in sample systems, including cells. The use of a caged ionophore allows the free ionophore to be produced within the sample with precise control, both temporally and spatially. By using focused laser illumination, the free ionophore can be generated at specific locations within a single cell or within a tissue, within the limits of the ability to focus the photolytic illumination.